Helicobacter pylori is a Gram-negative bacterium that is present in the stomach of at least half of the world's human population. In this niche, it persists for decades unless treated, and consistently induces gastric inflammation. Infection with this organism is a significant risk factor for the development of peptic ulcer disease, gastric carcinoma, and gastric lymphoproliferative diseases. The long-term objective of this project is to elucidate pathogenic mechanisms whereby H. pylori causes human disease, and to develop effective means for prevention and treatment of infection. In an effort to accomplish these goals, this proposal focuses investigation upon an important virulence determinant of H. pylori: the vacuolating cytotoxin. Although nearly all isolates contain a gene (vacA) encoding the cytotoxin, only about 50% produce vacuolating cytotoxin activity in vitro. Infection with cytotoxin-producing (tox+) strains is associated with an increased risk for peptic ulcer disease; moreover, administration of the purified cytotoxin intragastrically to mice induces gastric ulceration. In preliminary studies, it has been demonstrated that vacA structural gene sequences in tox+ strains differ substantially from those in tox- strains. In addition, it has been demonstrated that levels of vacA transcription are increased in tox+ strains compared to tox- strains. Finally, it has been demonstrated that the cytotoxin binds to epithelial cells and is internalized prior to inducing cytoplasmic vacuolation. The hypotheses of this proposal are (i) that the tox+ phenotype is dependent upon the presence of specific vacA structural gene domains which are absent from vacA homologs in tox- strains, (ii) that vacA transcription is regulated differently in tox+ and tox- strains, and (iii) that specific vacA domains or subunits are required for binding of the toxin to cells, intracellular trafficking, and induction of vacuolation. The specific aims are: to determine the basis for differences among H. pylori strains in levels of vacuolating cytotoxin activity, and to identify antigenic and functionally important domains of the cytotoxin. To accomplish the first objective, a series of chimeric vacA genes, derived in part from a tox+ strain and in part from a tox- strain, will be constructed and analyzed. In addition, a trans- acting vacA regulatory element will be sought. To accomplish the second objective, a series of recombinant vacA peptides will be synthesized, and polyclonal antisera prepared for these peptides. The antisera will be tested for the capacity to block binding and intracellular trafficking of the cytotoxin, as well as vacuole formation. In addition, the products of a series of vacA deletion mutants will be tested for functional activity. Finally, the regions of vacA that elicit an antibody response in humans will be determined by testing human sera for reactivity with the panel of recombinant vacA peptides. Understanding the structure and regulation of the vacuolating cytotoxin may aid in the development of strategies to prevent and treat H. pylori-associated gastroduodenal illnesses.